1. Field of the Invention
This invention relates to improvements in critical point dryers for sample preparation in electron microscopy and especially to a fully automatic critical point dryer.
Description of Prior Art
In order to examine biological specimens under a scanning electron microscope, they must be completely dried and coated with a thin conductive layer. It is important that the drying process be accomplished without disturbing the microstructure of the specimen to be examined. Normally this drying is accomplished in one of three manners depending upon the specimens's structure. Air drying by evaporation of the cellular water, while suitable for bacteria or other rigid structures, is detrimental to the structures of many specimens. The surface tension forces, which turn a grape into a raisin during the drying process, cause sufficient distortion in the cell structure of many specimens so as to render them useless. Sublimation or freeze-drying is useful only for very small specimens. Additionally, unless the lengthy technique is followed precisely structural damage from thermal expansion or ice crystal formation often results. The third method utilized is the phase transition or critical point drying which produces consistently reproducible results without the drawbacks of the preceding two methods.
In critical point drying the water contained in a specimen is gradually replaced by a dehydrating fluid such as ethanol or acetone. This maintains the three dimensional hydrated morphology of the structure under study. However, if the ethanol or acetone is now allowed to evaporate its surface tension forces would cause structural damage and destroy the specimen's usefulness and therefore the critical point drying method is utilized.
Critical point drying devices for sample preparation in electron microscopy are known in the art. The prior art critical point dryers utilize the technique of substituting a transitional fluid for the dehydrating fluid in the cell structure and then removing the transitional fluid. The critical point dryers heat and pressurize the specimen until above the critical pressure and critical temperature.
The critical temperature is defined as the temperature above which a gas cannot be liquefied by pressure alone. The critical pressure is the pressure that results when a substance exists as a gas and a liquid in equilibrium at the critical temperature. The critical point of a liquid is when its temperature and pressure are at or above the critical temperature and pressure and the densitites of the liquid phase and vapor phase are identical. This critical point is characterized by an absence of phase boundaries that normally exist between a liquid and its vapor at temperatures and pressures below the critical point. This absence of a phase boundary eliminates the boundary forces which exist when changing a liquid to a gas. It is precisely these boundary forces which cause the destruction of the extremely delicate specimens when changing its internal liquid to a gas below the critical point. Therefore, the solution which is applied in a critical point drying process is to remove the internal liquid from the specimen above its critical pressure and temperature to eliminate the boundary force destruction that would otherwise result.
Although all fluids have a characteristic critical point which should allow direct removal without the use of dehydrating or transition fluids, the critical point temperature and pressure of water is 374.2.degree. C and 218 atmospheres. Achieving these temperatures and pressures would cause severe damage to most bio-organic specimens and therefore a fluid having a lower critical temperature and pressure is normally substituted. Commonly, a dehydrating fluid is used which is miscible with water, for example, ethanol or acetone as an intermediate stage between the specimen containing water and a specimen containing transitional fluid.
Typically, and in the prior art dryers, the transitional fluid commonly used is carbon dioxide (CO.sub.2) because it is easy to use, more economical, less noxious and provides consistently better results than other transitional fluids. Its critical temperature and pressure is 31.degree. C and 1,072 psi, respectively, thus reducing the potential for destruction of the specimen structure.
The known instruments and apparatuses for critical point drying of specimens include, of course, a specimen chamber to which is connected a supply of the transitional fluid, CO.sub.2, with various regulating valves, temperature gages and a means for heating the chamber. A skilled technician must carefully control the application, heating, pressurizing and removal of the transitional fluid, thus requiring not only time but constant attention. Occasionally, automatic controls for one of the multi-steps in handling a specimen has been provided, but to the applicant's knowledge there has been no fully automated critical point dryer which eliminates the need for constant operator attention.